1.5 x 106 compound treated parasite per well were lysed using 1 mM EDTA, 40 mM Hepes pH 7.5, 50 mM Tris-HCl pH 7.5, 2% Triton-X100 and protease inhibitors cocktail (Sigma). are limited by their toxicity, administration in endemic countries and treatment resistance. Therapies against infectious diseases typically rely on targeting one or more components of the parasite that are not present in humans to ensure the best possible therapeutic window. In this case we aimed at targeting the trypanothione reductase (TR), one enzyme that synthesize the reduced trypanothione a key molecule for preserving the parasite redox balance. This enzyme does not exist in humans that have glutathione instead of trypanothione. Past attempts to identify novel inhibitors of this target has failed to generate drug-like molecules. To overcome this limitation we employed a recent, higher quality, TR activity assay to test a collection of compounds previously reported to be active against these parasites. This approach led to the identification and validation of a new chemotype with a unique mode of inhibition of TR. MSH4 This chemical series is usually a drug-like starting point, in fact its core (spiro) is present in drugs approved for human use. Introduction spp. and spp. are parasites belonging to Tryanosomatidae family that includes important pathogens of both human and animal. It is estimated that about 25 million people worldwide are affected by these two protozoa [1]. In particular is responsible for neglected pathologies such as chronic and acute human African trypanosomiasis (HAT), also known as sleeping sickness [2]. HAT is usually endemic in sub-Saharan Africa, with 70 million people at risk of infection. Late-stage HAT is usually usually fatal if untreated. The current therapeutic approaches for the treatment of trypanosomiases such as HAT include the use of organoarsenic compounds (e.g. melarsoprol) or diamidine derivatives (e.g. pentamidine)[3,4]. More recently, oral compounds like fexinidazole or oxaboroles have come to fruition.[5] Fexinidazole, in particular is the first all-oral drug targeting both early and late stages of sleeping sickness. [6] However, the inherent toxicity of some of these treatments, together with the dissemination of drug resistance [2,7,8], and the limited central nervous system (CNS) penetration to treat late stage HAT has limited the employment of these molecules, highlighting the need for new therapies to treat parasite infections [2,8]. In recent years, new biochemical pathways essential for parasite survival have emerged as you possibly can therapeutic target for the development of new drugs against trypanosomiases [9]. Of these, Trypanothione reductase (TR), a flavoenzyme that reduces trypanothione (TS2) CGS 35066 to its T(SH)2 form [10], CGS 35066 is usually of significant interest. T(SH)2 and TR represent a major defense system against oxidative stress for parasites, similar to the glutathione (GSH)Cglutathione reductase (GR) system found in humans. Despite the three-dimensional similarity between TR and GR, these enzymes recognize specific substrates (trypanothione vs. glutathione), suggesting the possibility to design specific and selective inhibitors of the parasite enzyme without off-target activity around the host[11]. cells lacking TR show an increased sensitivity to oxidative stress and limited virulent characteristics [12]. Targeting TR thus represents a viable approach to reduce Trypanosoma virulence. Several molecules have been characterized as TR inhibitors, such as polyamine, peptide derivate, benzimidazole, nitrobenzene derivate, quinazoline [13C19]. However, despite the large number of TR inhibitors reported in the literature, none of these series has reach drug development stages due to them being not drug-like.[20] These compound potency, toxicity and pharmacokinetics profiles [21] are often suboptimal and the large hydrophobic active site of TR [22] makes its inhibition by small molecules challenging. As a consequence the discovery and of new scaffolds able to inhibit TR activity is usually compulsory. In the present work we identify a new CGS 35066 inhibitor of TR (parasite and obtained.